Hair coloring composition comprising anti-hair antibodies immobilized on coloring materials, and hair coloring methods

ABSTRACT

A hair dye consisting of an anti-hair antibody immobilized on a high bulky coloring material. The coloring material may be a coloring substance such as an inorganic pigment, water-soluble coloring matter, or water-insoluble coloring matter, or a composite of the coloring substance and a macromolecular carrier. Since it provides a good and strong coloring capability specific to hair, the hair dye of the present invention will neither stain the skin nor cause skin irritation. Further, the touch and feel of the hair will be improved.

TECHNICAL FIELD

The present invention relates to hair dyes which are excellent incoloring, are resistant to color fading, do not produce color on skinbecause of their specific affinity to hair, do not irritate skin andimprove the tactile properties of hair.

BACKGROUND ART

For the past years, various methods of dyeing hair have been known inthe art. In one of the conventional methods, oxidation dyes arepermeated into hair in an alkaline condition, treated with oxidizingagents such as hydrogen peroxide or the like and then are polymerizedwithin the hair for developing color. Alternatively, acid dyes arepermeated into the hair for dyeing. There is also another temporarycoloring method in which coloring matter or pigment is adhered or fixedto hair using binders for developing colors.

However, those methods are known to have various disadvantages. Forexample, oxidation dyes pose problems such as damaged hair due to dyeingtreatment, allergic effects and irritation to skin, especially to mucusand eye. Although less irritating to skin, the acid dyes are often notsatisfactory in finish and disadvantageous in losing color when hair iswashed. Skin is sometimes stained by dyes when hair is dyed. Thetemporary coloring method does not provide a good tactile propertybecause of stickiness, brittleness or the like of applied binders. Inaddition, resistance to abrasion is generally not sufficient in thismethod.

Recently, antibodies have been used as cosmetics. For example, JapaneseLaid-open Patent Application No. 4-29912 discloses a method in whichantibodies are modified with functional molecules of dyes, perfumes,humectants and the like, and then said functional molecules aretransferred together with the antibodies to the desired place on accountof the specificity of the antibodies.

However, no testing is described about hair dyes in the above patentapplication though there is a description to the effect that an antibodymodified with phycopyrine protein (a fluorescent substance correspondingto a water-soluble natural dye) was applied to skin in an example of theabove patent. In addition, according to the above-mentioned method,there exist such problems that sufficient amounts of dyes required tocolor hair are hardly immobilized on the antibody and that too much loadof the dyes may inactivate the antibody. Thus, this method cannot be putinto practice.

On the other hand, a method has been known in which antibody-modifiedpigments are used as diagnostic reagents. However, in this method thepigments are used as a label only for the purpose of the temporaryconfirmation of the existence of targeted substances, and are notintended to color the targeted substances as dyes. Accordingly, nosignificant investigations have been conducted on coloring efficiencyand color durability (coloring strength) as dyes.

DISCLOSURE OF INVENTION

Therefore it is an object of this invention to provide hair dyes orcosmetics which are excellent in coloring, are resistant to colorfading, do not produce color on skin because of their specific affinityto hair, do not irritate skin and improve the tactile properties ofhair, and pretreatment agents for strengthening hair dyeing effects andmethods for dyeing hair with high effectiveness.

The afore-mentioned object is accomplished by the present inventionwhich comprises:

(1) Hair dyes made of high-bulky coloring materials on which anti-hairantibodies have been immobilized.

(2) Hair dyes for the secondary hair dyeing made of coloring materialson which antibodies having an immunoactivity to anti-hair antibodieshave been immobilized.

(3) Pretreatment agents for hair-dye antibodies which contain at leastone chemical agent selected from the group consisting of reducingagents, surfactants, alkaline substances and enzymes.

(4) Hair cosmetics or hair care products containing said hair dyes orsaid secondary hair dyes in an amount of 0.01-80% based on the totalweight.

(5) Hair cosmetics or hair care products containing said pretreatmentagents for hair-dye antibodies in an amount of 0.01-20% based on thetotal weight.

(6) A method for dyeing hair in which hair is treated with saidpretreatment agents for hair-dye antibodies and then is colored withsaid hair dyes.

(7) A method for dyeing hair in which hair is treated with the abovehair dye and then additionally colored with the above secondary hairdye.

The present invention will be explained in detail hereinafter.

Anti-hair antibodies as used in the present invention are defined asantibodies having an immunoactivity to human hair, and possess amolecular diameter of up to 10 nm. These include antibodies againstvarious components that constitute the hair to be colored. Theseantibodies can be obtained by immunizing animals using as antigens hairkeratin protein, hair cuticle protein, hair matrix protein or fragmentsthereof. Further, these antibodies can be obtained by using animalnails, body hair, feathers, or extracts and fragments thereof asantigens. In view of differences among species, human hair from eitherthe head or the body is preferably used, and especially variouscomponents that constitute human hair are most preferable.

Those animals to be immunized can be selected from domestic animals thatinclude bovines, horses, sheep, goats, rabbits, chickens and the like.

Antibodies can be isolated from milk or colostrum, serum, or egg yolkproduced by these animals. The antibodies obtained from bovine milk orcolostrum, and egg yolk are preferable because they can be produced in alarge quantity.

The raw materials of. the antibody described above can be purified bymeans of various methods known in the art. For example, after lipid hasbeen removed by suitable methods, crude antigens can be isolated bypurification processes including ammonium sulfate fractionation, alcoholprecipitation, membrane separation or the like. If required,ion-exchange chromatography and gel-filtration chromatography can beused for further purification. Further, if required, affinitychromatography can be conducted for super purification in which theantigen used in immunization serves as ligand.

The cell fusion process can be conducted between antibody-producingcells and myeloma cells to produce monoclonal antibodies.

The antibodies thus obtained can be digested with enzymes such aspapain, pepsin and the like to produce antibody fragments in which theFc part of immuno-globulin has been severed. Further, the H-chain andL-chain obtained by reducing the antibody with 2-mercaptoethanol may beused.

According to the present invention, antibodies having an immuno-activityto anti-hair antibodies can be obtained by immunizing different speciesof animals using, as antigens, antibodies and fragments thereof derivedfrom the same animal as the animal immunized in order to obtain saidanti-hair antibodies, and possesses about the a same molecular size asthat of the anti-hair antibody. Herein these antibodies are referred toas "secondary antibodies". For example, in the case where anti-hairantibodies have been obtained from milk, those antibodies derived from abovine are used to immunize. animals other than the bovine, for example,rabbits and chickens. In addition, monoclonal antibodies can be usedthat have an immuno-activity to anti-hair antibodies. These secondaryantibodies can be purified in the same manner as described in the caseof anti-hair antibodies.

The antibodies thus obtained may be digested with enzymes such aspapain, pepsin and the like to produce antibody fragments in which theFc part of immuno-globulin has been severed. Further, the H-chain andL-chain obtained by reducing the antibody with 2-mercaptoethanol may beused.

In the present invention, said anti-hair antibodies and said secondaryantibodies may be collectively referred to as "antibody raw material".The antibody raw materials can be immobilized on coloring materialshaving at least the same size as them, that is, a substance-constitutingunit size of at least about 10 nm. The "substance-constituting unitsize" means a "molecular size" in the case of water-soluble coloringsubstances while it means a "particle size" of materials finely groundin the form of use or fine powder materials in the case ofwater-insoluble coloring substances.

In this specification, those substances having a maximumsubstance-constituting unit size in diameter of at least about 10 nm aredefined as "high-bulky" while those substances having asubstance-constituting unit size less than that diameter are defined as"low-bulky". The substance-constituting unit size of this order can bemeasured directly by means of electron-microscopic observation, andfurther can be calculated by applying molecular models in the case ofwater-soluble coloring substances. Furthermore, "coloring materials"should be understood to refer to coloring substances themselves such asinorganic pigments and organic coloring matter and also to compositesubstances comprising these coloring substances and macromolecularcarriers. Then, "coloring substances" mean low-bulky water-solublecoloring matter, high-bulky water-insoluble coloring matter andinorganic pigments.

In case the coloring materials mean coloring substances themselves,these coloring substances are high-bulky water-insoluble coloring matterand inorganic pigments, and in case the coloring materials are coloringsubstance/macromolecular carrier composites, the coloring materials caninclude low-bulky water-soluble coloring matter. In either case, thecoloring materials are required to be of high-bulk, that is, of asubstance-constituting unit size of at least 10 nm. On the other hand,the coloring materials of low-bulk, that is, of a substance-constitutingunit size of less than 10 nm can not provide a sufficient degree ofcoloring. Nevertheless, the low-bulky coloring materials may be used forimmobilizing secondary antibodies. The substance-constituting unit sizeis desired not to exceed 100 μm. Sizes greater than 100 μm are likely toprovide significant negative effects on the tactile properties of dyedhair, and accordingly are considered to be undesirable.

The following table shows the coloring materials of the presentinvention and the coloring substances that constitute said coloringmaterials. ##STR1##

Coloring substances used in the present invention include water-solublecoloring matter such as water-soluble dyes and natural water-solublecoloring matter; water-insoluble coloring matter such as water-insolubledyes, lakes, organic pigments and water-insoluble natural coloringmatter; and inorganic pigments such as titanium oxides such as titanblack and titan white; iron oxide and magnetic particles. Among thesecoloring matters, inorganic pigments and water-insoluble coloring matterare preferred because of lower irritation to skin. Further, theinorganic pigments are preferred because they enable anti-hairantibodies to firmly bind to hair.

In addition, the inorganic pigments are preferably treated with couplingagents or other suitable agents to provide organic functional groups onthe surface thereof.

The substance-constituting unit size or particle size of water-insolublecoloring matter and inorganic pigments should be larger than 10 nm toprovide coloring substances of high-bulk. The size of the inorganicpigments is preferably in the range of 0.01-6 μm.

Organic pigments applicable to the present invention include BrilliantFast Scarlet, Permanent Red F5R, Hansa Orange, Hansa Yellow,Phthalocyanine Blue, Lithol Rubine BCA, Lake Red C, Lake Red CBA, LitholRed, Lithol Red CA, Lithol Red BA, Lithol Red SR, Deep Maroon, ToluidineRed, Permaton Red, Permanent Orange, Benzidine Orange G, BenzidineYellow G and the like.

High-bulky, water-insoluble dyes applicable to the present inventioninclude Alizarine Cyanine Green F, Quinizarin Green SS, indigo,Carbanthrene Blue, Alizurine Purple SS, medicated Scarlet Blue, Oil RedXO, Orange SS, Yellow AB, Yellow OB, Sudan Blue, Rhodamine B stearate,tetrachloro-tetrabromofluoresceine, tetrabromofluoresceine, Sudan III,dibromofluoresceine, diiodefluoresceine, fluoresceine, Quinoline YellowSS and the like.

Lakes applicable to the present invention include Naphthol BlueBlack•aluminum salt, Alizurol Purple•aluminum Lake salt and the like.

Water-soluble natural coloring matter applicable to the presentinvention includes shisonin, crocin, Safrole Yellow, Cacao coloringmatter, brazilin, and the like.

Water-insoluble natural coloring matters applicable to the presentinvention include capsanthin, riboflavin, chlorophyll, and the like.

Low-bulky Water-soluble dyes include Pyranine Conc, Light Green SFYellow, Patent Blue NA, Patent Blue CA, Alphazurin FG, Resorcine Brown,Violamine R, Ponceau 3R, Ponceau R, Ponceau SX, Fast Red S, Orange I,Polar Yellow 5G, Naphthol Yellow S, Metanil Yellow, Fast Light Yellow3G, Naphthol Green B, Guinea Green B, Alizrol Purple, Naphthol BlueBlack, Amaranth, Erythrosine, New Coccine, phloxine B, Rose Bengale,Acid Red, Tartrazine, Sunset Yellow FCF, Fast Green FCF, Brilliant BlueFCF, Indigo Carmine, Lithol Rubine B, Lithol Red, Rhodamine B, RhodamineB acetate, Fast Acid Magenta, Eosine YS, Eosine YSK, Phloxine BK, RoseBengale K, Orange II, Erythrocine Yellow NA, uranine, Uranine K,Quinoline Yellow WS and the like.

In the present invention, in the case where said coloring.materials arehigh-bulky coloring substances themselves including water-insolublecoloring matter, inorganic pigments and mixtures thereof, anti-hairantibodies can be directly immobilized on said coloring materials, thatis, high-bulky coloring substances. The ratio of anti-hair antibodies tocoloring substances preferably ranges 1:5-1:100 by weight. Ratiosgreater than 1:5 are likely to provide insufficient coloring capability.On the other hand, with ratios less than 1:100, no substantial increasein coloring effect is attained, even with an increase in an amount ofcoloring substances, and especially when the coloring substances arewater-soluble coloring matter, the anti-hair antibodies are prone to bedeactivated.

Further, said anti-hair antibodies can be immobilized on high-bulky,coloring substances/macromolecular carrier composite coloring materialsobtained by combining high-bulky coloring substances includinghigh-bulky water-insoluble coloring matter, inorganic pigments andmixtures thereof with said macromolecular carrier. In these instances,the ratio of said macromolecular carriers to said coloring substancespreferably ranges from 1:0.001-1:10 by weight. The secondary antibodiescan be immobilized directly on the coloring substances, and the ratio ofsaid secondary antibodies to said coloring substances preferably rangesfrom 1:0.1-1:100 by weight. In addition, the secondary antibodies can beimmobilized on the coloring substances/macromolecular carrier compositecoloring materials obtained by combining said coloring substances withsaid macromolecular carriers, and the ratio of said macromolecularcarriers to said coloring substances preferably ranges from 1:0.001-1:10by weight. Ratios greater than 1:0.001 are likely to provideinsufficient coloring capability. On the other hand, with ratios lessthan 1:10, no substantial increase in coloring effect is attained, evenwith an increase in an amount of coloring substances.

In the case where said coloring materials are high-bulky coloringsubstances such as water-insoluble coloring matter and inorganicpigments, either anti-hair antibodies are immobilized directly on them,or said anti-hair antibodies are immobilized on coloring materials madeof high-bulky coloring substance/macromolecular carrier composites. Boththe above instances would provide a better affinity of an anti-hairantibody to hair as compared with an instance where said anti-hairantibodies are immobilized directly on low-bulky coloring substancessuch as water-soluble coloring matter. The first instance where saidanti-hair antibodies are immobilized directly on the high-bulkyinorganic pigments is preferable because of a stronger affinity to hair,and on the other hand the second instance where said anti-hairantibodies are immobilized via the macromolecular carriers on thecoloring substances is also preferable because of a significantimprovement in functionality. Further, in the case where anti-hairantibodies are immobilized on low-bulky coloring substances made ofwater-soluble coloring.matter, it is preferable to use coloringsubstance/macromolecular carrier composites.

The macromolecular carriers used in the present invention are dividedinto two categories: water-insoluble macromolecular carriers andwater-soluble macromolecular carriers. The former water-insolublemacromolecular carriers are advantageous in improvements in tensity,resiliency, tautness, slipperiness, collectivity and feel of hair, andin particular synthetic polymers are preferable because of significantimprovement in tensity, resiliency and tautness of hair. In addition tothese improvements, water-insoluble proteins can provide suppleness,water-insoluble polysaccharides can provide humid feeling, and liposomescan provide gloss. On the other hand, the latter water-solublemacromolecular carriers are advantageous in improving humid feeling,smoothness, flexibility, slipperiness and feel of hair.

The water-insoluble macromolecular carriers include synthetic polymers,water-insoluble proteins, water-insoluble polysaccharides, liposomes andthe like.

The synthetic polymers include polymers such as polystyrene,poly(α-methylstyrene), polyvinyltoluene, polychloromethylstyrene,polychlorostyrene, polyvinylchloride, polyvinylbromide,polyacrylonitrile, polymethacrylonitrile, polyethylacrylate,polyoctylacrylate, polyhydroxypropylacrylate, polybutylacrylate,polymethoxyethylacrylate, polyhydroxyethylacrylate, polylaurylacrylate,polyammoniumacrylate, polymethylmethacrylate, polyethylmethacrylate,polypropylmethacrylate, polybutylaminoethylmethacrylate,polyhydroxymethacrylic acid, polyvinylacetate, polyacrylic acid,polymethacrylic acid, polymaleic acid, polystyrene sulfonic acid,poly(2-acrylamide-2-methylpropane sulfonic acid), polyacrylamide,polymethacrylamide, poly[N-(2-hydroxypropyl) methacrylamide],poly(2-hydroxyethyl methacrylate), poly(glycerolmonomethacrylate),poly(2-oxyethyl acrylate), poly(2-oxyethyl methacrylate), polyethyleneglycol methacrylate, polyethylene, polypropylene, polybutene,polyisobutene and the like; copolymers thereof; polyurethanes;copolymers of polyurethane with silicone and the like; nylon beads andthe like. Further, those polymers obtained by reforming the surface ofthe afore-mentioned synthetic polymers also can be used.

Among these synthetic polymers, preferably included are the followinggroups: hydroxy, halogen, amino, carboxylic, aldehyde, and sulfonicacids. These polymers include polychlorostyrene, polychloromethylstyrene, polyacrylic acid, polymethacrylic acid, polymaleic acid,polystyrene sulfonic acid, poly(2-acrylamide -2-methylpropane sulfonicacid), poly[N-(2-hydroxypropyl) methacrylamide], poly(2-hydroxyethylmethacrylate), poly(glycerol monomethacrylate), poly(2-oxyethylacrylate), poly(2-oxyethyl methacrylate), polyethylene glycolmethacrylate and the like; copolymers thereof; and polymers to whichreactivity is provided by surface-reforming. Polystyrene and itsderivatives are preferred on account of availability and ease with whichparticle diameters can be controlled.

As to the water-insoluble proteins, there can be used any proteins thatare insoluble in neutral water, and any modified proteins that have beenturned water-insoluble by being polymerized by the cross-linkage ofwater-soluble proteins. They include fibroin, gelatin, collagen and soon.

The water-insoluble polysaccharides include cross-linked agarose,cross-linked dextran, chitin and the like.

As to lipids used as raw materials for liposomes, mention may be made ofphospholipids such as phosphatidylcholine, phosphatidylserine,phosphatidyl ethanolamine, and the like.

Particle forms of these water-insoluble macromolecular carriers are notparticularly limited in the present invention, and any form such asspheres and plates may be used. Preferably, spheres with a uniform grainsize are used. Further, the size is not particularly limited, andpreferably is in the range of an average diameter of 0.001-100 μm, andmore preferably 0.001-1 μm. The most preferred range is 0.05-0.7 μm withwhich size a high hair dyeing efficiency can be accomplished.

The ratio of the water-insoluble macromolecular carriers to the antibodyraw materials depends on the water-insoluble macromolecular carrier tobe used. However, in general 0.01-100 mg of the antibody raw material ispreferably immobilized on 1 g of the water-insoluble macromolecularcarrier. Less than 0.01 mg is likely to provide insufficient coloringcapability. On the other hand, with the addition of more than 100 mg, nosubstantial increase in coloring capability is attained.

As to the water-soluble macromolecular carriers, either natural orsynthetic ones can be used, which include polysaccharides and proteinsof natural origin and derivatives thereof.

Those polysaccharides include starch, amylose, amylopectin, pectin,carrageenan, mannan, galactan, sodium alginate, tragacanth gum, gumarabic and the like, of vegetable origin; dextran, pullulan, Curdlan,levan, glucan, succinoglucan, Xanthane Rubber and the like, ofmicroorganisum origin; and hyaluronic acid, chondroitin sulfate and thelike, of animal origin.

Those proteins include glue, gelatin, casein, collagen, fibroin and thelike.

The semi-synthetic polymers include viscose, methyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,carboxymethyl cellulose, solubilized starch, carboxymethyl starch,dialdehyde starch and the like.

The synthetic polymers include polyvinylalcohol, carboxyvinyl polymers,polysodiumacrylate, polyvinylpyrrolidone, polyethyleneoxide, polylysine,polyglutamic acid, polyaspartic acid and the like.

Any molecular form of these water-soluble macromolecular carriers,either of straight chain or branched chain can be used. Preferably,polymers with a uniform molecular weight distribution are used, andpreferably is in the range of an number-averaged molecular weight of10,000-2,000,000. In addition, the average substance-constituting unitsize or molecular size is preferred to be in the range of 0.01-5 μm.

The ratio of the water-soluble macromolecules to the antibody rawmaterials depends on the water-soluble macromolecule to be used.However, in general, 0.01 mg-1 g of the antibody raw material ispreferably immobilized on 1 g of the water-soluble macromolecule.

Hair dyes of the present invention can be produced using the antibodyraw materials according to the following methods, but methods of theinvention are not limited to those methods described herein.

According to the present invention, in order to immobilize coloringsubstances such as water-insoluble coloring matter and inorganicpigments directly on the antibody raw materials, physical adsorption andchemical binding are utilized.

Physical adsorption can occur while the water-insoluble coloring matterand inorganic pigments are mixed with the antibody raw materials.

Chemical binding can occur when the waterinsoluble coloring matter orinorganic pigments possess organic functional groups. In the case ofinorganic pigments that usually lack organic functional groups, it isnecessary to introduce organic functional groups on the surface of thepigment. Treatments using coupling agents and silicone treatment can beused for the introduction of organic functional groups on the surface ofthe pigment. The coupling agents include silane coupling agents,titanate coupling agents, aluminate coupling agents, zirco-aluminatecoupling agents and the like. Amino-modified silicone andcarboxyl-modified silicone and the like are used for the siliconetreatment.

For example, 3-aminopropyl triethoxy silane can be reacted to introduceamino groups to the pigment surface. Reaction of the amino group withglutaric anhydride or succinic anhydride can transform the amino groupto a carboxyl group. Further, 3-mercaptopropyl triethoxy silane canreact to introduce an SH group to the pigment surface.

These organic functional groups can be combined with the antibody rawmaterials according to any well-known methods. If the organic functionalgroup is an amino group, then glutaraldehyde is used to combine it whilea carboxyl group can be combined with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (abbreviated as EDChereinafter). If an SH group is combined, cross-linking reagents asexemplified by N-succinimidyl-3-(2-pyridyl dithio)propionate(abbreviated as SPDP hereinafter) can be used.

According to the present invention, in order to support coloringsubstances such as water-soluble coloring matter, water-insolublecoloring matter, inorganic pigments and the like on the water-insolublemacromolecular carriers or water-soluble macromolecular carriers,physical and chemical methods are utilized.

The physical methods include an adsorption method in which the coloringsubstances are adsorbed on the water-insoluble macromolecular carriers,an internal addition method in which the coloring substances are addedto the water-insoluble macromolecular carrier during its manufacture, aninclusion method in which the coloring substances are included withinthe water-insoluble macromolecular carriers, and a polymerization methodin which coloring matter precursors are polymerized within thewater-insoluble macromolecular carriers.

The chemical binding also can immobilize the coloring substances iffunctional groups exist in the water-insoluble or water-solublemacromolecular carriers. The functional groups include amino groups,carboxyl groups, aldehyde groups, hydroxyl groups, thiol groups, and thelike. Polysaccharides can be used per se, but may be chemicallymodified, if required. For example, polysaccharides can be oxidized withmetaperiodate acid and the like to form aldehyde groups. Diamines can bereacted with the aldehyde groups to introduce an amino group whileε-aminocaproic acid can be reacted with the aldehyde groups to introducea carboxyl group. In addition, activation reagents such as cyanuricchloride and cyanogen bromide can be reacted with polysaccharides toform activated polysaccharides.

According to the present invention, in order to immobilize antibodies onwater-insoluble or water-soluble macromolecular carriers, physicaladsorption and chemical binding are utilized.

The functional groups of antibody raw materials that are used for thechemical binding include amino groups, carboxyl groups, aldehyde groups,thiol groups, polysaccharide chains and the like, which can beimmobilized in the same manner as in the binding between coloringsubstances and carriers. For example, when polysaccharide are used forbinding, the saccharide chain portions can be oxidized withmetaperiodate and the like to form aldehyde groups, which are thenreacted with amino groups of the carriers to form Schiff bases, thusresulting in immobilization. Thiols can be reacted with SPDP reagentsfor immobilization.

In manufacturing the aforementioned antibody raw materials immobilizedcoloring materials, ratios of the antibody raw materials to coloringmaterials and ratios of the macromolecular carriers to the coloringsubstances can be changed so that their ratios in theantibody-immobilized coloring materials thus formed can be adjusted toobtain desired ratios of their components.

Hair dyes comprising the antibody raw materials-immobilized coloringmaterials that have been obtained as described above can be put intopractice as solutions in appropriate solvents, such as aqueous solution,or as dispersions, or as dried products that are obtained by means offreeze-drying or spray-drying.

The hair dyes of the present invention possess sufficient amounts ofcoloring substances to be required for coloring so that theyspecifically react and combine with hair to effect coloring. Further, inthe case of hair dyes using macromolecular carriers, there are achievedextraordinary improvements in functions of hair. In the case where themacromolecular carriers are water-insoluble macromolecular carriers,there will be improvements in tensity, resiliency, tautness,slipperiness, collectivity and tactile properties of the hair. Inaddition to these improvements, water-insoluble proteins can providesuppleness, water-insoluble polysaccharides can provide humid feel, andliposomes can provide gloss. On the other hand, the water-solublemacromolecular carriers are advantageous in improvement in humid feel,smoothness, suppleness, slipperiness and tactile properties of the hair.Hair dyes in which antibody raw materials have been immobilized directlyto water-soluble macromolecular carriers or inorganic pigments accordingto the present invention will have a stronger bonding to hair ascompared with conventional coloring matter-direct bonded antibodies.

The antibody raw materials-immobilized coloring materials of the presentinvention can be incorporated into conventional hair cosmetics insuitable amounts to provide useful hair cosmetics. Hair cosmetics thatcontain only conventional hair dyes, when applied to hair, may color thevery scalp in a certain case, or in another case, do not have sufficientconcentrations due to fear of skin troubles, resulting in poor coloringcapability. Thus, it has been quite troublesome to repeat hair dyeingoperations until a desired degree of coloring is reached since only asmall degree of coloring is attained with a single hair dyeingoperation. However, the hair cosmetics of the present invention canprovide satisfactory hair dyeing effects with only a single action ofdyeing while causing no staining on the scalp at all, since the hair dyecontains a sufficient amount of anti-hair antibody immobilized coloringmaterials which bond specifically to hair.

The hair cosmetics of the present invention can be prepared bycompounding said hair dyes together with a conventional substrate forhair cosmetics. Forms suitable for such hair cosmetics include shampoo,rinse, styling foam, hair conditioner, hair pack, hair cream, hairliquid, hair tonic, permanent wave reagents, hair manicures and thelike.

The content of hair dye in the hair cosmetics of the present inventionmay be determined suitably based on the types of cosmetics, types ofcoloring substances, types and sizes of carriers and the like. Ingeneral, based on the total weight of the cosmetics, its content ranges0.01-80% by weight with the balance being conventional substrates forhair cosmetics.

Reducing agents used for pretreating the hair dye antibody of thepresent invention include sodium hydrogen sulfite, sodium sulfite,potassium pyrosulfate, sodium pyrosulfate, sodium thiosulfate,L-cysteine, thioglycolic acid, 2-mercaptoethanol, 2-mercaptopropionicacid, thioglycerol and the like. Surfactants include SDS (sodium dodecylsulfonate) and the like. Alkaline substances include ammonia,monoethanolamine and the like. Enzymes include papain, trypsin and thelike.

Pretreatment of hair is conducted by treating hair with an aqueoussolution containing said pretreatment agents. Further, in thispretreatment step, ultrasonic application can increase pretreatmentefficiency.

The obtained pretreatment agents for hair dye antibodies can becompounded together with conventional substrates for hair cosmetics toproduce the hair cosmetics of this invention.

The content of the pretreatment agent for hair dye antibodies in thehair cosmetics of the present invention can be determined suitably basedon the types of cosmetics and types of pretreatment agents and the like.In general, based on the total weight of the cosmetics, its contentranges 0.01-20% by weight with the balance being conventional substratesfor hair cosmetics.

In order to color hair with the hair dyes of this invention or with thehair cosmetics containing said hair dyes, it is sufficient only to applythe hair dyes made of anti-hair antibodies or the hair cosmeticscontaining said hair dyes. In addition, if hair is treated in advancewith the pretreatment agents of this invention for hair dye antibodiesor with the hair cosmetics containing said pretreatment agents, thenhair coloring efficiency will be increased greatly, and thispretreatment is preferable. Furthermore, if hair is colored additionallywith the secondary hair dyes made of secondary antibodies or antibodieshaving an immuno-activity to anti-hair antibodies or with the cosmeticscontaining said secondary hair dyes, then hair coloring efficiency alsowill be increased greatly. Further, hues of hair after having been dyedcan be changed by using coloring substances of types different from thetype used the previous time.

THE BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be explained using the following examples,but are not limited to these examples. Percentages as described in theexamples are based on weight unless specifically noted.

Example 1 Anti-Keratin Antibody-Immobilized Titan Black Hair Dyes

(1) Preparation of Antigens

5 g of male healthy hair and 5 g of female healthy hair were mixed, andwashed with a 2% sodium polyoxyethylene laurylether sulfate (3 E.O.)aqueous solution. The washed healthy hair was agitated under N₂ bubblingin a 2.5 l 0.2M Tris-HCl buffer solution (pH 9.2) containing 8M urea and0.2M 2-mercaptoethanol at 50° C. for one hour, and then ground with aTEFLON® homogenizer. The above extraction procedure was repeated and theobtained extract was subjected to 1,000 × g centrifugation for 30minutes to remove impurities and to obtain a hair keratin antigenextract. 200 g of monoiodide acetic acid (which was dissolved in 760 mlsolution containing 400 g tris) was then added to the extract for thereaction conducted while stirring in darkness at room temperature forone hour. 7 ml of 2-mercaptoethanol was added to stop the reaction. Theresulting mixture was sufficiently dialyzed against distilled water. A 5μm filter was used to remove insolubles to obtain 6 l of an aqueoussolution of hair keratin antigen. To 4 parts by volume of this solutionwas added 1 part by volume of a 0.25M sodium acetate buffer solution(adjusted to pH 4.2 with acetic acid), and hair keratin was precipitatedat the isoelectric point. The precipitate thus obtained was thencentrifuged with a 10,000× g force for 10 minutes. The supernatant wasremoved to collect the precipitate which was then dissolved inphysiological salt water. A 0.2 μm filter was used to remove germs fromthe solution which was then concentrated using ultrafiltration membranesto obtain purified hair keratin antigen (2.6 g as protein).

(2) Preparation of Anti-Keratin Antibody

(Immunization Into Cow)

The protein concentration of said purified hair keratin antigen solutionwas adjusted at 20 mg/ml with physiological salt water. The solution andcomplete Freund's adjuvant were mixed at a volume ratio of 1:1 toprepare a water-in-oil emulsion. Two heads of pregnant Holstein cows 2months before parturition were injected with 5.0 ml each of saidemulsion subcutaneously in the neck. Subsequently, with an interval of10 days an, emulsion that had been prepared with incomplete Freund'sadjuvant and that contained the same amount of antigen as in thefirst-time immunization was injected subcutaneously or intramuscularlyfor immunization. (1st-3rd immunization: hypodermic injection, 4-5thimmunization: intramuscular injection).

(Collection and purification of antibody)

Colostrum was collected from the cows immunized as described above forthree days immediately after parturition. A cream separator was used toseparate the fat layer of the colostrum to obtain skimmed milk. From theskimmed milk thus obtained, fractional purification of antibodies wasconducted by the following procedure.

To the skimmed milk was added 0.1N HCl to adjust the pH at 4.5 toprecipitate casein. The precipitate was roughly filtered out. Thesupernatant was obtained through a continuous centrifugation at 2,500×g. The obtained supernatant was neutralized. Ammonium sulfate was addedto obtain a 33% saturated liquid from which the antibody was salted out.Precipitates were collected by a 2,500× g centrifugation, and dissolvedin a phosphate buffered saline solution (abbreviated as PBShereinafter). This ammonium sulfate salt-out procedure was repeated. Theresulting solution was dialyzed against a 10 mM PBS (pH 7.5). Thesolution was divided into 5 aliquots, each of which was applied to a 2 lDEAE cellulose column (DE-52, made by Whatman) that had beenequilibrated with said buffer solution. Unadsorbed proteins were washedaway by said buffer solution. Afterwards, the antibody was eluted withsaid buffer solution containing 50 mM NaCl, and the antibody fractions(200 g as antibody) were collected. The fractions had an antibody purityof 90% or higher. The fraction was divided into 5 aliquots, each ofwhich was provided to 400 ml affinity resin (Affi-Gel 15, Bio-Rad, Inc.)to which purified hair keratin had been conventionally bound. Anti-hairkeratin antibodies adsorbed to the affinity resin were eluted with a0.2M glycine HCl buffer solution (pH 2.5), and immediately pH adjustedto around 8 with a 3M tris solution. Then, the anti-hair keratinantibody (affinity-purified) that specifically binds to the hair keratinantigen was obtained. For simplicity, this antibody will be referred toas an anti-keratin antibody hereinafter.

(3) Preparation of Control Antibody

Antibodies were obtained from the colostrum of nonimmunized cows andwere purified by means of the same methods as described above to obtaincontrol antibodies. The control antibodies used in the comparativeexamples had been purified in a DEAE-cellulose column and had anantibody purity of 90% or more.

(4) Immobilization of Anti-Keratin Antibody on Titan Black

(Surface Modification of Titan Black)

1 g of Titan Black 10 S (average particle diameter of 0.025 μm, producedby Mitsubishi Metal Co.) was dispersed in 10 ml of distilled water usingultrasonic waves. To this dispersion liquid was added 75 μl of anaqueous 1% 3-aminopropyl triethoxysilane solution, and the resultingmixture was agitated at room temperature for 2 hours. While being heatedat 40° C., the mixture was dewatered under a vacuum in an evaporatorThen, 10 minutes' drying operation at 110° C. introduced amino groups onthe surface of the Titan Black.

(Reaction between Silane-treated Titan Black and Glutaric aldehyde)

80 mg of Titan Black to which an amino group had been introduced bysilane treatment was dispersed in 4 ml of distilled water usingultrasonic waves. To this dispersion liquid was added 4 ml of an aqueous0.25% glutaric aldehyde solution, and the resulting mixture was agitatedat room temperature for 2 hours. Then, the mixture was centrifuged at20° C., 10,000 rpm for 20 minutes, and then redispersed in 8 ml ofdistilled water. This washing procedure was repeated 5 times.

(Reaction of Titan Black with Anti-Keratin Antibody)

To 0.5 ml of a 1% Titan Black dispersion liquid treated with glutaricaldehyde was added 0.5 ml of an anti-keratin antibody solution that hadbeen adjusted to 10 mg/ml with PBS, and the resulting mixture wasagitated at 4° C. overnight. Then, the mixture was centrifuged at 20°C., 12,000 rpm for 20 minutes, and then redispersed in 0.5 ml of PBScontaining 0.1% bovine serum albumin (abbreviated as BSA hereinafter).Further, the mixture was centrifuged at 20° C., 12,000 rpm for 20minutes, and then redispersed in 0.5 ml of PBS containing 0.1% BSA, andafterwards incubated at room temperature for 1 hour. Then, to thismixture was added 10 μl of an aqueous 0.005M sodium borohydridesolution, and the resultant mixture was incubated at room temperaturefor 1 hour, and then centrifuged at 20° C., 12,000 rpm for 20 minutes.The mixture was then redispersed into 0.5 ml of PBS containing 0.1%TWEEN 20 (polyoxyethylene sorbitan monolaurate, 20 E.O.) and 0.1% BSA.On the hair dye thus obtained, it was found, 144 mg of the antibody per1 g of the Titan Black had been immobilized.

Comparative Example 1

Instead of the anti-keratin antibody produced in Example 1, the controlantibody produced in Example 1(3) was immobilized on Titan Black. ThisTitan Black was used to produce a hair dye which was designated asComparative Example 1. On the hair dye thus obtained, it was found, 243mg of the antibody per 1 g of the Titan Black had been immobilized.

Example 2 Anti-Keratin Antibody-Immobilized Titan Black Hair Dye(Reaction between Silane-treated Titan Black and Glutaric Anhydride)

25 mg of Titan Black that had been treated with silane in Example 1(4)was dispersed in 4 ml of an aqueous 0.1M sodium hydrogen carbonate.Then, the mixture was centrifuged at 20° C., 10,000 rpm for 20 minutes,and then redispersed in 4 ml of an aqueous 0.1M sodium hydrogencarbonate. This procedure was repeated 4 times. At the last step, themixture was redispersed in 0.5 ml of an aqueous 0.1M sodium hydrogencarbonate. To this mixture was added 50 μl of glutaric anhydride aqueoussolution (57 mg/ml), and the resulting mixture was agitated at roomtemperature for 1 hour to effect reaction. Then, the mixture wascentrifuged at 20° C., 10,000 rpm for 20 minutes, and then redispersedin 4 ml of an aqueous 0.1M sodium hydrogen-carbonate. This procedure wasrepeated 3 times. At the last step, the mixture was redispersed in 0.5ml of an aqueous 0.1M sodium hydrogen carbonate.

To this mixture was added 50 μl of glutaric acid anhydride aqueoussolution (57 mg/ml), and the resulting mixture was agitated for reactionat room temperature for 1 hour. Then, the mixture was centrifuged at 20°C., 10,000 rpm for 20 minutes, and afterwards the reactant was removed.The, the mixture was redispersed in 4 ml of an aqueous 0.1M sodiumhydrogen carbonate, and centrifuged at 20° C., 10 000 rpm for 20 minutesfor washing the Titan Black. This operation was repeated 2 times. At thelast step, the mixture was redispersed in 0.5 ml of an aqueous 0.1Msodium hydrogen carbonate. Again, to this mixture was added 50 μl ofgluralic anhydride aqueous solution (57 mg/ml), and the resultingmixture was agitated for reaction at room temperature for 1 hour. Then,the mixture was centrifuged at 20° C., 10,000 rpm for 20 minutes, andafterwards the reactant was removed. The, the mixture was redispersed in4 ml of distilled water, and centrifuged at 20° C, 10,000 rpm for 20minutes for washing the Titan Black. This operation was repeated 4times. At the last step, the mixture was redispersed in 2.5 ml ofdistilled water. These treatments provided Titan Black to which acarboxyl group had been introduced.

(Reaction of Titan Black with Anti-Keratin Antibody)

The pH of a 1% Titan Black aqueous dispersion liquid to which a carboxylgroup had been introduced with gluralic anhydride treatment was adjustedto be 5. To 0.5 ml of this dispersion liquid was added 0.5 ml of a0.002M EDC aqueous solution, and the resulting mixture was agitated forreaction at room temperature for 2 hours. Then, the mixture wascentrifuged at 20° C., 12,000 rpm for 20 minutes, and then redispersedin 0.5 ml of distilled water. To this EDC-activated Titan Black wasadded 0.5 ml of the anti-keratin antibody solution that had beenadjusted to 10 mg/ml with PBS as in Example 1(2), and then the mixturewas agitated at 4° C. overnight. Afterwards, the resultant mixture wascentrifuged at 20° C., 12,000 rpm for 20 minutes, and then redispersedinto 0.5 ml of PBS containing 0.1% BSA. Again, this was centrifuged at20° C., 12,000 rpm for 20 minutes, and then redispersed in 0.5 ml of PBScontaining 0.1% BSA., and afterwards incubated at room temperature for 1hour. Then, this was centrifuged at 20° C., 12,000 rpm for 20 minutes,and then redispersed in 0.5 ml of PBS containing 0.1% TWEEN 20 and 0.1%BSA. On the hair dye thus obtained, it was found, 166 mg of the antibodyper 1 g of the Titan Black had been immobilized.

Comparative Example 2

Instead of the anti-keratin antibody produced in Example 2, the controlantibody produced in Example 1(3) was immobilized on Titan Black. ThisTitan Black was used to produce a hair dye which was designated asComparative Example 2.

Example 3 Anti-Keratin Antibody-Immobilized Titan Black Hair Dye(Surface Modification of Titan Black)

1 g of Titan Black 10S (produced by Mitsubishi Metal Co.) was dispersedin 20 ml of distilled water using ultrasonic waves. To this dispersionliquid was added 15 μl of a zircoaluminate coupling agent havingcarboxyl groups (type C, produced by MANCHEM, Inc.), and the resultingmixture was agitated at room temperature for 2 hours. While being heatedat 40° C., the mixture was dewatered under a reduced pressure in anevaporator. Then, 10 minutes of a drying operation at 110° C. produced aTitan Black treated with the zircoaluminate coupling agent. Thistreatment could provide a Titan Black on the surface of which a carboxylgroup had been introduced.

(Reaction of Titan Black with Antibody)

The Titan Black on the surface of which a carboxyl group has beenintroduced by means of the zircoaluminate coupling treatment wasdispersed into distilled water at a concentration of 1%. To 0.5 ml ofthis dispersion liquid was added 0.5 ml of a 0.002M EDC aqueoussolution, and the resulting mixture was agitated for reaction at roomtemperature for 2 hours. Then, the mixture was centrifuged at 20° C.,12,000 rpm for 20 minutes, and then redispersed in 0.5 ml of distilledwater. To this EDC-activated Titan Black was added 0.5 ml of theanti-keratin antibody that had been adjusted to 10 mg/ml with PBS as inExample 1(2), and then the mixture was agitated at 4° C. overnight.Afterwards, the resultant mixture was centrifuged at 20° C., 12,000 rpmfor 20 minutes, and then redispersed in 0.5 ml of PBS containing 0.1%BSA. Again, this was centrifuged at 20° C., 12 000 rpm for 20 minutes,and then redispersed in 0.5 ml of PBS containing 0.1% BSA., andafterwards incubated at room temperature for 1 hour. Then, this wascentrifuged at 20° C., 12,000 rpm for 20 minutes, and then redispersedin 0.5 ml of PBS containing 0.1% TWEEN 20 and 0.1% BSA. On the hair dyethus obtained, it was found, 60 mg of the antibody per 1 g of the TitanBlack had been immobilized.

Comparative Example 3

Instead of the anti-keratin antibody produced in Example 3, the controlantibody produced in Example 1(3) was immobilized on Titan Black. ThisTitan Black was used to produce a hair dye which was designated asComparative Example 3.

Example 4 Anti-Keratin Antibody-Immobilized Carbon Black Hair Dye(Reaction of Carbon Black with Antibody)

60 g of Carbon Black MA 100 (an average particle diameter of 0.022 μm,produced by Mitsubishi Chemicals Co.) was dispersed in 10 ml of a 0.05 %TWEEN 20 solution using ultrasonic waves. To 10 ml of this carbon blackdispersion liquid was added 10 ml of a 0.02M EDC aqueous solution, andthe resulting mixture was agitated for reaction at room temperature for2 hours. Then, the mixture was centrifuged at 20° C., 10 000 rpm for 20minutes to remove unreacted EDC. The EDC-activated carbon black wasredispersed in 10 ml of the 0.05 % TWEEN 20 solution. That is, 0.5 ml ofa 0.6 %arbon black dispersion liquid was charged into a tube to whichalso 0.5 ml of the anti-keratin antibody that had been adjusted to aprotein concentration of 1.0 mg/ml with PBS as in Example 1(2) wasadded, and then the mixture was agitated at 4° C. overnight. Aftercompletion of the reaction, the resultant mixture was centrifuged at 20°C., 10,000 rpm for 20 minutes, and then the carbon black thus obtainedwas redispersed in 1.0 ml of PBS containing 0.1% BSA. Further, this wascentrifuged at 20° C,, 10,000 rpm for 20 minutes, and then redispersedin 1.0 ml of PBS containing 0.1% BSA., and afterwards incubated at roomtemperature for 1 hour. Then, this was centrifuged at 20° C., 10,000 rpmfor 20 minutes, and then redispersed in 1.0 ml of PBS containing 0.1%TWEEN 20 and 0.1% BSA. Finally, a 0.6% carbon black dispersion liquid towhich an anti-keratin antibody had bound was obtained.

Comparative Example 4

Instead of the anti-keratin antibody produced in Example 4, the controlantibody produced in Example 1(3) was immobilized on carbon black. Thiscarbon black was used to produce a hair dye which was designated asComparative Example 4.

Example 5 Anti-Keratin Antibody-Immobilized Carbon Black Hair Dye(Surface Modification of Carbon Black)

To 2.5 g of Carbon Black No. 4 (an average particle diameter of 0.025μm, produced by Degussa Co.) was added 50 ml of distilled water orisopropyl alcohol, and the mixture was dispersed using ultrasonic waves.To this dispersion liquid was added 16.5 μl of a zircoaluminate couplingagent (type C, produced by MANCHEM, Inc.), and the resulting mixture wasagitated at room temperature for more than 2 hours. While being heatedat 40° C. or higher, the mixture was dehydrdated under a reducedpressure in an evaporator. Then, the product was dried at 110° C. for 15minutes. This process could provide a carbon black on the surface ofwhich a carboxyl group had been introduced.

(Reaction of Carbon Black with Antibody)

Carbon Black No. 4 to which a carboxyl group had been introduced wasdispersed in distilled water so that its concentration was 1%. To 0.5 mlof this carbon black dispersion liquid was added 0.5 ml of a 0.02M EDCaqueous solution, and the resulting mixture was agitated for reaction atroom temperature for 2 hours. Then, the mixture was centrifuged at 20°C., 14,500 rpm for 20 minutes to remove unreacted EDC. The EDC-activatedcarbon black was redispersed in 0.5 ml of distilled water for use ofcombination with an antibody. That is, 0.5 ml of a 1.0% carbon blackdispersion liquid was charged into a tube to which also 0.5 ml of theanti-keratin antibody that had been adjusted to a protein concentrationof 10 mg/ml with PBS as in Example 1(2) was added, and then the mixturewas agitated for reaction at 4° C. overnight. After completion of thereaction, the resultant mixture was centrifuged at 20° C., 14,500 rpmfor 20 minutes, and then the carbon black thus obtained was redispersedin 1.0 ml of PBS containing 0.1% BSA. Further, this was centrifuged at20° C., 14,500 rpm for 20 minutes, and then redispersed in 0.5 ml of PBScontaining 0.1% BSA, and afterwards incubated at room temperature for 1hour. Then, this was centrifuged at 20° C., 14,500 rpm for 20 minutes,and then redispersed in 0.5 ml of PBS containing 0.1% TWEEN 20 and 0.1%BSA. Finally, a 1.0% carbon black dispersion liquid to which ananti-keratin antibody had bound was obtained.

Comparative Example 5

Instead of the anti-keratin antibody produced in Example 5, the controlantibody produced in Example 1(3) was immobilized on carbon black. Thiscarbon black was used to produce a hair dye which was designated asComparative Example 5.

Example 6 Anti-Keratin Antibody-Immobilized Iron Oxide Hair Dye (SurfaceModification of Iron Oxide)

Water-insoluble polyacrylic acid polymer was dissolved in toluene toprepare a 5% polyacrylic acid polymer solution. To this solution wasadded 25% by weight of iron oxide (an average particle diameter of0.02×0.06 μm), and the mixture was dispersed well using ultrasonicwaves. After complete dispersion, the mixture was then heated forevaporation of toluene to obtain the iron oxide coated with polyacrylicacid polymers.

(Reaction of Iron Oxide with Antibody)

The polymer coated iron oxide was dispersed in distilled water at asolids concentration of 1%. Afterwards, HCl was used to adjust the pH to5. To 1 ml of this dispersion liquid was added 1.0 ml of a 0.01M EDCaqueous solution, and the resulting mixture was agitated for reaction atroom temperature for 2 hours. Then, the mixture was centrifuged toremove supernatant, and redispersion operations were conducted withadditional uses of 1.0 ml of PBS. To this was added 1.0 ml of theanti-keratin antibody that had been adjusted to a concentration of 1.0mg/ml with PBS as in Example 1(2), and then the mixture was agitated at4° C. overnight. After centrifugation, the supernatant was removed, andredispersion operations were conducted with additional uses of 1.0 ml ofPBS containing 0.1% BSA. Afterwards, the mixture was incubated at roomtemperature for 1 hour to conduct blocking by BSA. Further, aftercentrifugation, redispersion was conducted with the addition of 1.0 mlof PBS containing 0.1% TWEEN 20 and 0.1% BSA. Finally, iron oxides towhich anti-keratin antibodies had been chemically bound were obtained.

Comparative Example 6

Instead of the anti-keratin antibody produced in Example 6, the controlantibody produced in Example 1(3) was immobilized on iron oxides. Theiron oxides were used to produce a hair dye which was designated asComparative Example 6.

Example 7 Anti-Keratin Antibody-Immobilized Colored Latex Hair Dye

Water-insoluble carboxy-modified-polystyrene latex (an average particlediameter of 0.19 μm, produced by Japan Synthetic Rubber Co.) on which ared coloring matter had been adsorbed was dispersed in distilled waterat a solids concentration of 1%. Afterwards, HCl was used to adjust thepH to 5. To 1 ml of this latex aqueous dispersion was added 1.0 ml of a0.01M EDC aqueous solution, and the resulting mixture was agitated atroom temperature for 2 hours. Then, the mixture was centrifuged toremove supernatant, and redispersion operations were conducted withadditional uses of 1.0 ml of PBS. To this was added 1.0 ml of theanti-keratin antibody that had been adjusted to a concentration of 1.0mg/ml with PBS as in Example 1(2), and then the mixture was agitated at4° C. overnight. After centrifugation, the supernatant was removed, andredispersion operations were conducted with additional uses of 1.0 ml ofPBS containing 0.1% BSA. Afterwards, the mixture was incubated at roomtemperature for 1 hour to conduct unreacted site blocking by BSA.Further, after centrifugation, redispersion was conducted with theaddition of 1.0 ml of PBS containing 0.1% TWEEN 20 and 0.1% BSA.Finally, a colored latex to which anti-keratin antibodies had beenchemically bound were obtained. On the hair dye thus obtained, it wasfound, 50-80 mg of the antibody per 1 g of the colored latex had beenimmobilized.

Comparative Example 7

Instead of the anti-keratin antibody produced in Example 7, the controlantibody produced in Example 1(3) was combined with the colored,water-insoluble latex. The latex thus obtained was used to produce ahair dye which was designated as Comparative Example 7. On the hair dyethus obtained, it was found, that 50-80 mg of the antibody per 1 g ofthe colored latex had been immobilized.

Example 8 Anti-Keratin Antibody-Immobilized Colored Latex Hair Dye

Water-insoluble carboxy-modified-polystyrene latex (an average particlediameter of 0.19 μm) on which red coloring matter had been adsorbed wasdispersed in distilled water at a solids concentration of 1%. To 1 ml ofthis dispersion was added 1.0 ml of the anti-keratin antibody that hadbeen adjusted to a concentration of 1.0 mg/ml with PBS as in Example1(2), and then the mixture was agitated at 4° C. overnight. Aftercentrifugation, the supernatant was removed, and a redispersionprocedures was conducted with additional uses of 1.0 ml of PBScontaining 0.1% BSA. Further, procedures of centrifugation andredispersion were repeated, and then redispersion was conducted with theaddition of 1.0 ml of PBS containing 0.1% TWEEN 20 and 0.1% BSA.Finally, a colored latex on which anti-keratin antibodies had beenimmobilized by means of physical adsorption was obtained. On the hairdye thus obtained, it was found, 50-80 mg of the antibody per 1 g of thecolored latex had been immobilized.

Comparative Example 8

Instead of the anti-keratin antibody produced in Example 8, the controlantibody produced in Example 1(3) was adsorbed to the colored,water-insoluble latex. The latex thus obtained was used to produce ahair dye which was designated as Comparative Example 8. On the hair dyethus obtained, it was found that 50-80 mg of the antibody per 1 g of thecolor latex had been immobilized.

Example 9 Anti-Keratin Antibody-Immobilized Colored Latex Hair Dye

Water-insoluble carboxy-modified-polystyrene latex (an average particlediameter of 0.15 μm) on which Carbon Black MA 100 as a black pigment hadbeen included was dispersed in distilled water at a solids concentrationof 1%. Afterwards, the pH was adjusted to 5 by HCl. To 1 ml of thislatex aqueous dispersion was added 1.0 ml of a 0.01M EDC aqueoussolution and the resultant mixture was agitated at room temperature for2 hours. After centrifugation, the supernatant was removed, andredispersion operations were conducted with additional uses of 1.0 ml ofPBS. This was admixed with 1 ml of the anti-keratin antibody that hadbeen adjusted to a concentration of 1.0 mg/ml with PBS as in Example1(2), and then was agitated at 4° C. overnight. After centrifugation,the supernatant was removed, and redispersion operations were conductedwith additional uses of 1.0 ml of PBS containing 0.1% BSA. Then, thesolution was incubated at room temperature for 1 hour followed byblocking of unreacted sites with BSA. Further, after centrifugation,redispersion was conducted with the addition of 1.0 ml of PBS containing0.1% TWEEN 20 and 0.1% BSA. Finally, a colored latex to whichanti-keratin antibodies had been bound by means of a chemical bond wasobtained.

Comparative Example 9

Instead of the anti-keratin antibody produced in Example 9, the controlantibody produced in Example 1(3) was combined with the colored,water-insoluble latex. The latex thus obtained was used to produce ahair dye which was designated as Comparative Example 9.

Example 10 Anti-Keratin Antibody-Immobilized Colored Latex Hair Dye

Water-insoluble carboxy-modified-polystyrene latex (an average particlediameter of 0.15 μm) on which Carbon Black MA 100 as a black pigment hadbeen included was dispersed in distilled water at a solid concentrationof 1%. 1 ml of this dispersion liquid was admixed with 1 ml of theanti-keratin antibody that had been adjusted to a protein concentrationof 1 mg/ml with PBS as in Example 1(2), and then the mixture wasagitated at 4° C. overnight. After centrifugation, the supernatant wasremoved, and redispersion operations were conducted with additional usesof 1.0 ml of PBS containing 0.1% BSA. Further, after operations ofcentrifugation and redispersion were repeated, redispersion wasconducted with the addition of 1.0 ml of PBS containing 0.1% TWEEN 20and 0.1% BSA. Finally, a colored latex on which anti-keratin antibodieshad been immobilized by means of physical adsorption was obtained.

Comparative Example 10

Instead of the anti-keratin antibody produced in Example 10, the controlantibody produced in Example 1(3) was adsorbed on the colored,water-insoluble latex. The latex thus obtained was used to produce ahair dye which was designated as Comparative Example 10.

Comparative Example 11

A hair dye was prepared by combining the antibodies as prepared inExample 1(2) directly with red coloring matter (water-soluble coloringmatter) in an equimolar amount without water-insoluble macromolecularcarriers.

Comparative Example 12

A hair dye was prepared by combining the antibodies as prepared inExample 1(2) directly with red coloring matter (water-soluble coloringmatter) in an excessive molar amount without water-insolublemacromolecular carriers.

Test Example 1 Agglutination Test

The hair-keratin antigens produced in Example 1(1) were adjusted withPBS at a 100 μg concentration. The hair-keratin solution was mixed withthose hair dyes of Examples 1-10 and Comparative Examples 1-12 on anequal amount basis, and whether or not agglutination occurred after 1minute was judged. The results are shown in Table 1. In this table, thesign "-" denotes that no agglutination was observed while the sign "+"denotes that agglutination was clearly observed. Further, the sign "±"denotes that it was difficult to determine whether or not agglutinationoccurred.

                                      TABLE 1    __________________________________________________________________________    Examples                  Comparative Examples    1       2 3 4 5 6 7 8 9 10                              1 2 3 4 5 6 7 8 9 10                                                  11                                                    12    __________________________________________________________________________    Results of          + + + + + + + + + + - - - - - - - - - - + -    judgment    __________________________________________________________________________

In the hair dyes of Examples 1-12 on which antikeratin antibodies wereimmobilized, agglutination was observed to indicate that the immobilizedantibodies maintained an immunoactivity against the antigens. Inaddition, it was found that, in Comparative Example 11 where theantibody was combined directly with the water-soluble coloring matter,an immunoactivity against the antigens was maintained while, inComparative Example 12 where the antibody was combined with theexcessive amount of the water-soluble coloring matter, theimmunoactivity had lost.

Test Example 2 Difference in Color of Hair Dye

The hair dyes produced in Example 7 and Comparative Examples 11 and 12were diluted so that each antibody concentration was the same as eachother. Then, color differences among each hair dye was evaluated torepresent the results in Table 2. In this table, the sign "-" denotesthat no red color was observed while the sign "+" denotes that the redcolor was clearly observed Further, the sign "±" denotes that it wassomewhat observed.

                  TABLE 2    ______________________________________                    Difference in color    ______________________________________    Example 7         +    Comparative Example 11                      -    Comparative Example 12                      ±    ______________________________________

The hair dye in Comparative Example 11 was hardly red in spite of thefact that the hair dye was the same antibody concentration as that ofthe antibody in Example 7 where the red color was clearly recognized.Further, in Comparative Example 12 where the antibody was combined withthe excessive amount of the water-soluble coloring matter, theimmunoactivity against antigens had lost as shown in Table 1. Thus thishair dye of the Comparative Example 12 was not suitable.

Test Example 3 Hair Dye Test

The hair dyes produced in Examples 1-10 and Comparative Examples 1-12were diluted with PBS containing 0.1% TWEEN 20 and 0.1% BSA, so thatconcentrations of the anti-hair antibody-immobilized coloring materials(=hair dyes) were all 0.1%. Then, in 1 ml of each hair dye was immerseda bundle of human grey hair (0.15 g), which was rotated at roomtemperature for 1 hour. Then, the hair bundle was washed while shook inphysiological salt water containing 0.02% TWEEN 20, and then wasair-dried. The degree of coloring was visually evaluated. The sign ◯denotes that the hair has been colored, and the sign A denotes that thehair has been somewhat colored. The sign x represents that the hair hasnot been colored at all. The results are summarized in Table 3.

                                      TABLE 3    __________________________________________________________________________    Examples                  Comparative Examples    1       2 3 4 5 6 7 8 9 10                              1 2 3 4 5 6 7 8 9 10                                                  11                                                    12    __________________________________________________________________________    Results of          ◯            ◯              ◯                ◯                  ◯                    ◯                      ◯                        ◯                          ◯                            ◯                              X X X Δ                                      Δ                                        X X X X X X X    coloring    hairs    __________________________________________________________________________

As can be seen from Table 3, the grey hair was indeed colored with thehair dyes of Examples 1-10 where the anti-keratin antibodies were used.On the other hand, the hair was not sufficiently colored in ComparativeExample 1-10 where the control antibody was used and also in ComparativeExamples 11 and 12 where water-soluble coloring matter was directlycombined with anti-keratin antibodies.

Test Example 4 Durability Test after Hair Dyeing

Using the hair dyes prepared in Examples 4, 5, 7, and 8, bundles of hairthat had been colored in the same manner as in Test Example 3 underwentthe following shampoo and brushing treatments. With these treatments,color durability was evaluated as follows.

(Shampoo Treatment)

Colored hair bundles were washed by hand in 10% aqueous solution of acommercially available shampoo, and then rinsed with running waterfollowed by air-drying. The degree of coloring was visually evaluated.

(Brushing Treatment)

Colored hair bundles were brushed with a pig-hair brush, and the degreeof coloring was visually evaluated.

The Results of each evaluation are represented in Table 4. It should benoted that only the shampoo treatment was applied with respect to hairdyes produced in Examples 4 and 5. In Table 4, an increasing number ofthe sign "+" indicates a higher degree of coloring. For the shampootreatment, the first time application caused a slight color fading inhair dyes produced in Examples 4 and 5, but substantially no color fadewas observed after the second time application, thus indicating thatgreater durability against shampoo treatment was demonstrated. Inparticular, hair dyes produced in Example 7 showed a high durability.For the brushing treatment, those hair dyes produced both in Examples 7and 8 demonstrated a high durability.

                  TABLE 4    ______________________________________    Number of repeats of Number of repeats of    shampoo treatment    brushing    0           1      2      3    0    100  300  1000    ______________________________________    Example 4            +++     ++     ++   ++    Example 5            +++     ++     ++   ++    Example 7            ++      ++     ++   ++   ++   ++   ++   +    Example 8            ++      +      -    -    ++   ++   +    +    ______________________________________

Example 11 Anti-Keratin-immobilized Hair Dye

Water-insoluble polymethyl methacrylate latex (100 μm) to which a bluecoloring matter had been adsorbed was combined with the anti-keratinantibodies of Example 1(2) in the same manner as in Example 2 to preparehair dyes.

Example 12 Anti-Keratin-immobilized Blue Dextran Hair Dye

2 g of CIBACRON BLUE 3GA (Sigma, Inc.), and 1 g of DEXTRAN T-40(molecular weight of 40,000, average molecular size of about 100 nm,water-soluble, produced by Pharmacia Co.) were dissolved in 90 ml ofdistilled water. Agitated at 60° C. for 30 minutes, then 9.88 g ofsodium chloride was added and agitated at 60° C. for 1 hour. Temperaturewas raised to 80° C., and 0.875 g of sodium carbonate was added andagitated 80° C. for 2 hours. Unreacted CIBACRON BLUE was removed bygelfilter chromatography using Sephadex-25. Then, after oxidizing withsodium metaperiodate overnight, the solution thus obtained was subjectedto dialysis against deionized water, and freeze-dried.

This freeze-dried CIBACRON BLUE-combined DEXTRAN T-40 was dissolved in a20 mM boric acid buffer solution containing 0.14M sodium chloride toreact with the anti-keratin antibody of Example 1(2) at 4° C. for 24hours. Then, the resultant solution was reduced with hydrogenated sodiumborate at 4° C. for 2 hours to obtain antibodies that had beenimmobilized on CIBACRON BLUE via dextran. On the hair dye thus obtained,it was found, 0.48 g of the CIBACRON BLUE per 1 g of dextran had beenimmobilized. In addition, about 0.5 g of the antibody per 1 g of dextranhad been immobilized.

Comparative Example 13

Instead of the anti-keratin antibody produced in Example 12, the controlantibody produced in Example 1(3) was immobilized on water-soluble bluedextran. This blue dextran was used to produce a hair dye which wasdesignated as Comparative Example 13.

The hair dyes produced in Example 12 and Comparative Example 13 werediluted with PBS containing 0.1% TWEEN 20 and 0.1% BSA, so thatconcentrations of the antibody were 0.1 mg/ml. Then, into this solutionwas immersed a bundle of human grey hair at room temperature for a wholeday and night. Then, the hair bundle was washed in physiological saltwater containing 0.02% TWEEN 20. The degree of coloring was observed. Asa result, it was found that, only in the case of Example 12, the greyhair had been colored. In this case it was demonstrated that this hairdye was resistent to color fading and that many functions such astensity, resiliency, tautness, slipperiness, collectivity and tactileproperties of hair had been improved.

Example 13 Anti-Keratin-immobilized Fluorescent Dextran Hair Dye(Preparation of Hair Dye)

DEXTRAN T-40 (water-soluble, produced by Pharmacia Co.) was oxidizedwith sodium metaperiodate for a whole day and night. After oxidation,the solution thus obtained was subjected to dialysis against deionizedwater to obtain oxidized dextran. An excessive amount of hexamethylenediamine was added to the oxidized dextran to react at 4° C. for 24hours, followed by a reduction treatment with hydrogenated sodium borateat 4° C. for 2 hours. Unreacted hexamethylene diamine was removed bygel-filtration chromatography using Sephadex G-25 (produced by PharmaciaCo.) to obtain dextran to which amino groups had been introduced.

The afore-mentioned dextran to which amino group had been introduced andfluoresceine isothiocyanate (abbreviated as FITC hereinafter) (producedby Sigma Inc.) were reacted in a 0.5M carbonic acid buffer solution (pH9.5) at 4° C. for 6 hours. Unreacted FITC was removed by gel-filterchromatography using Sephadex G-25, and then the solution was reactedwith formaldehyde in the presence of hydrogenated sodium borate to blockthe remaining amino groups. The mixture was dialyzed against distilledwater. Thus, dextran to which FITC had been introduced was obtained.

The afore-mentioned dextran to which FITC had been introduced was againoxidized with sodium metaperiodate. Then, the resultant solution wasreacted to combine with amino groups of the anti-keratin antibodiesproduced in Example 1(2), thus obtaining antibodies that had beenimmobilized via dextran on FITC.

Comparative Example 14

The FITC-conjugated, water-soluble dextran without antibodies producedin Example 13 was designated as Comparative Example 14.

Comparative Example 15

Instead of the anti-keratin antibody produced in Example 13, the controlantibody produced in Example 1(3) was immobilized on the dextran. Thedextran thus obtained was designated as Comparative Example 15.

Comparative Example 16

To 2 ml of the anti-keratin antibody (10 mg/ml) produced in Example 1(2)and adjusted with PBS was added 0.2 ml of a 0.5M carbonic acid buffersolution (pH 9.5). Then, to the resultant solution was again added 0.2ml of FITC (1.0 mg/ml) dissolved in the same buffer solution to react inthe dark at 4° C. for 6 hours. After centrifugation, the reactedsolution was applied to a Fast Desalting FPLC (available from PharmaciaCo.) equilibrated with a 0.005M PBS (pH 8.4) to remove unreacted FITC.DEAE Sepharose Fast Flow (available from Pharmacia Co.), was chargedwith about 5 ml of the resultant solution which was then washed with a0.005M PBS (pH 8.4). The FITC-labeled conjugated antibodies adsorbed tothe gel were eluted successively with a 0.005M PBS (pH 8.4)•(the firsteluent), a 0.1M PBS (pH 6.4)•(the second eluent), and a 1.0M PBS (thethird eluent) in this order. The third eluate fraction was collected anddialyed against physiological salt water. Thus, FITC-conjugatedanti-keratin antibodies were obtained, and designated as ComparativeExample 16.

Comparative Example 17

Instead of the anti-keratin antibody produced in Comparative Example 16,the control antibody produced in Example 1(3) wast conjugated with FITC,which was designated as Comparative Example 17.

Test Example 5 Evaluation of Binding Force to Hair

Bundles of hair colored in the same manner as in Test Example 3 usinghair dyes produced in Examples 4 and and Comparative Example 16 weretreated for one hour in eluents (1)-(4) shown in Table 5 and evaluatedhow the degreeof hair coloring would change. Visual observation wasapplied to hair dyes in Examples 4 and 7. On the other hand, afluorescence microscope was used to observe hair colored with hair dyesin Comparative Examples 16

                  TABLE 5    ______________________________________    Eluents    ______________________________________    (1) 3M Sodium thiocyanate (pH 7.4)    (2) 6M guanidine HCI (pH 3)    (3) 8M urea (pH 7)    (4) 50% ethylene glycol (pH 11)    ______________________________________

The results of each evaluation are represented in Table 6. In Table 6,increasing number of the sign "+" indicates higher degree of coloring.Because of interaction between carbon black and hair, binding to hair inExample 4 was stronger than in Comparative Example 16, thus making itshair dye more resistant to color fading. In other words, it has beenfound that antibodies immobilized directly on inorganic pigments canbind more strongly with hair than antibodies immobilized directly onwater-soluble coloring matter. Further, it has been found that evenwater-soluble coloring matter can be strongly bound to hair byimmobilizing antibodies via water-insoluble macromolecular carriers onwater-soluble coloring matter as evidenced in Example 7.

                  TABLE 6    ______________________________________             Before  Eluents             elution (1)    (2)      (3)  (4)    ______________________________________    Example 4  ++        ++     ++     ++   ++    Example 7  ++        +      +      ++   -    Comparative               +         -      -      -    -    Example 16    Example 7  ++        +      +      ++   -    Comparative               +         -      -      -    -    Example 16    ______________________________________

Test Example 6 Sensuality Test after Hair Coloring

Sensuality tests were conducted on bundles of hair colored in the samemanner as in Test Example 3 using hair dyes produced in Examples 4 and 7and Comparative Example 16. 10 testing professionals judged how tensity,resiliency, tautness, slipperiness, collectivity and tactile propertiesof hair had changed over controls of hair bundles before coloring. Theresults of the judgment are shown in Table 7. Sensuality of the hairdyes of Examples 4 and 7 was improved as compared with that of Example16. In particular, sensuality had been greatly improved in the hair dyesproduced using colored latex in Example 7.

                  TABLE 7    ______________________________________                      Example Comparative    Test items          4      7      Example 16    ______________________________________    Tensity  Number of testers                            6      10   6             responded positive             Number of testers                            4      0    4             responded neutral    Resiliency             Number of testers                            7      9    3             responded positive             Number of testers                            3      1    7             responded neutral    Tautness Number of testers                            8      8    2             responded positive             Number of testers                            2      2    8             responded neutral    Slipperiness             Number of testers                            7      6    4             responded positive             Number of testers                            3      4    6             responded neutral    Collectivity             Number of testers                            5      7    2             responded positive             Number of testers                            5      3    8             responded neutral    Tactile  Number of testers                            6      7    2    property responded positive             Number of testers                            4      3    8             responded neutral    ______________________________________

Test Example 7 Dyeing Test

Into each hair dye produced in Example 13 and Comparative Examples 14-17was immersed a bundle of human grey hair at room temperature for a wholeday and night. Then, the hair bundle was washed with physiological saltwater containing 0.02% TWEEN 20. Fluorescent light was applied tocolored hair to judge whether or not a difference was made influorescence strength between before and after coloring treatments. Theresults are presented in Table 8.

Test Example 8 Color Fading Test

The bundle of hair colored in Example 7 was washed with a commerciallyavailable shampoo. Fluorescent light was applied to the colored hair tojudge whether or not a difference was made in fluorescence strengthbetween before and after coloring treatments. Color fading was decidedon the basis of the number of testing professionals who recognized thedifference in fluorescence strength. The results are presented in Table8.

Test Example 9 Sensuality Test

Professionals had judged how tensity, resiliency, tautness,slipperiness, collectivity and tactile properties of hair had changed ascompared with hair bundles before coloring. The results are alsopresented in Table 8.

The judgment on each test in Examples 7 and 8 was made by 10 testingprofessionals.

                                      TABLE 8    __________________________________________________________________________                                    Compar-                                         Compar-                                              Compar-                                                   Compar-                                    ative                                         ative                                              ative                                                   ative                               Example                                    Example                                         Example                                              Example                                                   Example                               13   14   15   16   17    __________________________________________________________________________    Test Example 7              Number of testers found                               10   3    3    4    1    (Coloring Test)              difference in fluorescence              Number of testers found no                               0    7    7    6    9              difference in fluorescence    Test Example 8              Number of testers found                               9    0    0    2    0    (Color Fading Test)              difference in fluorescence              Number of testers found no                               1    10   10   8    10              difference in fluorescence    Test Example 9              Dampishness                     Number of testers                               9    2    1    2    10    (Sensuality      responded positive    Test)            Number of testers                               1    8    9    8    10                     responded neutral              Smoothness                     Number of testers                               8    1    3    2    2                     responded positive                     Number of testers                               2    9    7    8    8                     responded neutral              Suppleness                     Number of testers                               8    0    1    2    1                     responded positive                     Number of testers                               2    10   9    8    9                     responded neutral              Slipperiness                     Number of testers                               9    2    3    3    2                     responded positive                     Number of testers                               1    8    7    7    8                     responded neutral              Tactile                     Number of testers                               7    1    2    2    1              property                     responded positive                     Number of testers                               3    9    8    8    9                     responded neutral    __________________________________________________________________________

Use of the hair dyes in Example 13 provided more fluorescent coloringmatter bound to hair bundles compared with the hair dyes in ComparativeExamples 14-17. It has been found that they are resistant to colorfading associated with washing. In addition, use of such hair dyesimproved tactile properties of hair itself.

Example 14 Rinse

The hair dye in Example 4 was formulated to prepare a rinse according toa method known in the art. The composition of the rinse having acoloring capability is shown in Table 9.

                  TABLE 9    ______________________________________                        (% by weight)    ______________________________________    Stearyl trimethyl ammonium chloride                          3.0    Cetanol               1.5    Glycerine monostearate                          1.5    1,3-butylene glycol   5.0    Liquid paraffin       2.0    Perfume               proper quantity    Hair dye of Example 4 5.0    Refined water         Balance    ______________________________________

The rinse thus prepared had been used continuously for one month.Evaluation covered coloring of grey hair, stains on faces and hands,color fading due to shampoo, itch and eruption of the scalp, andsensuality. As the results, the rinse prepared in Example 14 was foundthat it did color grey hair well without any stains on face or hand. Nocolor fading was observed after shampooing. There was neither itch noreruption on the scalp. The sensuality of the hair also had beenimproved.

Example 15 Rinse

The hair dye in Example 7 was formulated to prepare a rinse according toa method known in the art. The composition of the rinse having acoloring capability is shown in Table 10.

                  TABLE 10    ______________________________________                        (% by weight)    ______________________________________    Stearyl trimethyl ammonium chloride                          3.0    Cetanol               1.5    Glycerine monostearate                          1.5    1,3-butylene glycol   5.0    Liquid paraffin       2.0    Perfume               proper quantity    Hair dye of Example 7 5.0    Refined water         Balance    ______________________________________

The rinse thus prepared had been used continuously for one month.Evaluation covered coloring of grey hair, stains on faces and hands,color fading due to shampoo, itch and eruption of the scalp, andsensuality. As the results, the rinse prepared in Example 15 was foundto dye well grey hair without any stains on face or hand. No colordegradation was observed after shampooing. There was neither itch noreruption on the scalp. The sensuality of the hair also had beenimproved. In addition, the same effects were recognized on those rinsesthat had been formulated with the hair dyes produced in Examples otherthan Examples 4 and 7.

Example 16 Rinse

The hair dye in Example 12 was formulated so as to have a compositionshown in Table 11. Thus, a was obtained having a coloring capability.

                  TABLE 11    ______________________________________    Stearyl trimethyl ammonium chloride                           3.0    Cetanol                1.5    Glycerine monostearate 1.5    1,3-butylene glycol    5.0    Liquid paraffin        2.0    Perfume                proper quantity    Hair dye of Example 12 5.0    Refined water          Balance to total 100    ______________________________________

After shampooing, grey hair was treated with said rinse. As a result,the grey hair was colored so that they could not seen distinctively.Further, it was found that the rinse treatment could improve tactileproperties of hair.

Example 17 Pretreatment with Alkali

A bundle of human white hair was treated in a 10% ammonia aqueoussolution for 30 minutes while ultrasonic waves were applied. Aftertreatment, the bundle was washed thoroughly with deionized water toobtain an alkali-pretreated hair bundle.

Example 18 Pretreatment with Surfactant

A bundle of human white hair was treated in a 1% SDS aqueous solutionfor an hour while ultrasonic waves were applied. After treatment, thebundle was washed thoroughly with deionized water to obtain anSDS-pretreated hair bundle.

Example 19 Pretreatment with L-Cystine

A bundle of human grey hair was treated in a 0.1M sodium carbonatebuffer solution (pH 9) containing 0.2M L-cystine at 30° C. for 10minutes. After treatment, the bundle was washed thoroughly withdeionized water to obtain an L-cystine-pretreated hair bundle

Example 20 Pretreatment with Thioglycolic Acid

A bundle of human grey hair was treated in a 0.1M sodium carbonatebuffer solution (pH 9) containing 0.2M thioglycolic acid at 30° C. for10 minutes. After treatment, the bundle was washed thoroughly withdeionized water to obtain a thioglycolic acid-pretreated hair bundle

Example 21 Pretreatment and Hair Dyeing

The human grey hair pretreated in Examples 17-20 was colored with thehair dyes of Examples 2, 4 and 7 according to the method of Test Example3.

Test Example 10 Coloring Efficiency Enhanced by Pretreatment

The hair dyed after the pretreatment in Example 21 was compared with theone dyed without any pretreatment. The results are shown in Table 12. Ithas been found that coloring efficiencies increased with pretreatment bymeans of alkali, SDS, L-cystine, and thioglycolic acid.

                  TABLE 12    ______________________________________           Pretreatment agents for coloring antibody                     Example  Example                                     Example                                            Example    Hair dyes             None    17       18     19     20    ______________________________________    Example 2             ◯                     ⊚                              ⊚                                     ⊚                                            ⊚    Example 4             ◯                     ⊚                              ⊚                                     ⊚                                            ⊚    Example 7             ◯                     ⊚                              ⊚                                     ⊚                                            ⊚    ______________________________________

Example 22 Shampoo

Thioglycolic acid as a pretreatment agent was compounded in a shampooaccording to a method known in the art. The shampoo composition is shownin Table 13.

                  TABLE 13    ______________________________________                        (% by weight)    ______________________________________    Polyoxyethylene lauryl ether sodium                          15.0    sulfate (2 E.O.)    Coconut oil fatty acid diethanolamide                          5.0    Ethylene glycol distearate                          1.5    Sodium benzoate       0.2    EDTA                  0.2    Coloring matter       proper quantity    Perfume               proper quantity    Thioglycolic acid     8.0    Refined water         Balance    ______________________________________

Test Example 11 Coloring Efficiency Enhanced by Pretreatment(Practicality Test)

With the cooperation of 3 volunteers who had distinct grey hair, a halfhead test was conducted. That is, a half of the head was treated withthe shampoo of Example 22 while the other half of the head was treatedwith a shampoo that had the same composition as that of Example 22except for thioglycolic acid. They all used the hair dye-formulatedrinse of Example 14.

As a result of one week's continuous use, grey hair, with the use of theshampoo of Example 22, was colored better than through the use of theother shampoo, and no grey hair had been distinctively seen on theformer half head.

Example 23 Rabbit Anti-Bovine IgG Antibody-Immobilized Titan Black HairDye

A rabbit was immunized with bovine IgG, and a rabbit anti-bovine IgGantibody was obtained. According to the same method as in Example 2, therabbit anti-bovine IgG antibody was immobilized on Titan Black. Thisrabbit anti-bovine IgG antibody-immobilized Titan Black was demonstratedto bind specifically to bovine IgG in such a agglutination test asconducted in Test Example 1.

Example 24 Multi-step Dyeing

A bundle of human white hair, which had been colored with a hair dyecomposed of anti-keratin antibody-immobilized Titan Black (Example 2),was again colored with a hair dye composed of the rabbit anti-bovine IgGantibody-immobilized Titan Black that had been produced in Example 23,according to the same method as in Test Example 3.

Test Example 12 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the additional coloring in Example 24, it has beenindicated that the human grey hair, which had been colored with a hairdye comprising anti-keratin antibody-immobilized Titan Black, could befurther richly colored as illustrated in Table 14. In the table, anincreasing number of the sign "+" indicates a higher degree of coloring.

Example 25 Rabbit Anti-Bovine IgG Antibody-Immobilized Carbon Black HairDye

According to the same method as in Example 5, the rabbit anti-bovine IgGantibody was immobilized on carbon black. This rabbit anti-bovine IgGantibody-immobilized carbon black was demonstrated to bind specificallyto bovine IgG in such a agglutination test as conducted in Test Example1.

Example 26 Multi-step Dyeing

A bundle of human grey hair, which had been colored with a hair dyecomprising anti-keratin antibody-immobilized carbon black (Example 5),was again colored with a hair dye composed of the rabbit anti-bovine IgGantibody-immobilized carbon black that had been produced in Example 25,according to the same method as in Test Example 3.

Test Example 13 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the multi-step dyeing in Example 26, it has beenindicated that the human grey hair, which had been colored with a hairdye comprising anti-keratin antibody-immobilized carbon black, could befurther richly colored as illustrated in Table 14.

Example 27 Rabbit Anti-Bovine IgG Antibody-Immobilized Red Latex HairDye

According to the same method as in Example 7, the rabbit anti-bovine IgGantibody was immobilized on red latex (water-insoluble). This rabbitanti-bovine IgG antibody-immobilized carbon black was demonstrated tobind specifically to bovine IgG in such a agglutination test asconducted in Test Example 1.

Example 28 Multi-step Dyeing

A bundle of human grey hair, which had been colored with a hair dyecomprising anti-keratin antibody-immobilized red latex (Example 7), wasagain colored with a hair dye comprising the rabbit anti-bovine IgGantibody-immobilized red latex that had been produced in Example 27,according to the same method as in Test Example 3.

Test Example 14 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the multi-step dyeing in Example 28, it has beenindicated that the bundle of human grey hair, which had been coloredwith a hair dye comprising anti-keratin antibody-immobilized red latex,could be further richly colored as illustrated in Table 14.

Example 29 Rabbit Anti-Bovine IgG Antibody-Immobilized Black Latex HairDye

According to the same method as in Example 9, the rabbit anti-bovine IgGantibody was immobilized on black latex (water-insoluble). This rabbitanti-bovine IgG antibody-immobilized carbon black was demonstrated tobind specifically to bovine IgG in such a agglutination test asconducted in Test Example 1.

Example 30 Multi-step Dyeing

A bundle of human grey hair, which had been colored with a hair dyecomprising anti-keratin antibody-immobilized black latex (Example 9),was again colored with a hair dye comprising the rabbit anti-bovine IgGantibody-immobilized black latex that had been produced in Example 27,according to the same method as in Test Example 3.

Test Example 15 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the multi-step dyeing in Example 30, it has beenindicated that the bundle of human grey hair, which had been coloredwith a hair dye comprising anti-keratin antibody-immobilized blacklatex, could be further richly colored as illustrated in Table 14.

Example 31 Rabbit Anti-Bovine IgG Antibody-Immobilized Blue Dextran HairDye

According to the same method as in Example 12, the rabbit anti-bovineIgG antibody was immobilized to blue dextran

Example 32 Multi-step Dyeing

A bundle of human grey hair, which had been colored with a hair dyecomprising anti-keratin antibody-immobilized blue dextran (Example 12),was again colored with a hair dye comprising the rabbit anti-bovine IgGantibody-immobilized blue dextran that had been produced in Example 31,according to the same method as in Example 12.

Test Example 16 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the multi-step dyeing in Example 32, it has beenindicated that the bundle of human grey hair, which had been coloredwith a hair dye comprising anti-keratin antibody-immobilized bluedextran, could be further richly colored as illustrated in Table 14.Thus, it has been demonstrated that plural repetitive dyeing treatmentswere effective in order to increase the degree of coloring the hair thathad been already colored in Example 12.

                  TABLE 14    ______________________________________    Primary Hair Dye               Secondary Hair Dye                              Degree of coloring    ______________________________________    Example 2  Example 23     +++    Example 2  --             ++    Example 5  Example 25     +++    Example 5  --             ++    Example 7  Example 27     +++    Example 7  --             ++    Example 9  Example 29     +++    Example 9  --             ++    Example 12 Example 31     ++    Example 12 --             +    ______________________________________

Examples 33 and 34 Multi-step Dyeing

A bundle of human grey hair that had been colored with a hair dyecomprising anti-keratin antibody-immobilized Titan Black (Example 2),and a bundle of human grey hair that had been colored with a hair dyecomprising anti-keratin antibody-immobilized carbon black (Example 5),were each colored again with a hair dye comprising the rabbitanti-bovine IgG antibody-immobilized carbon black that had been producedin Example 25 and a hair dye comprising the rabbit anti-bovine IgGantibody-immobilized Titan Black that had been produced in Example 23,receptively, according to the same method as in Test Example 3.

Test Example 17 Change in Coloring Efficiency and Hue by Multi-stepDyeing

As a result of the multi-step dyeing in Examples 32 and 34, it has beenindicated that the bundle of human grey hair, which had been coloredwith a hair dye comprising anti-keratin antibody-immobilized TitanBlack, could not only be further richly colored with a hair dyecomprising rabbit anti-bovine IgGa antibody-immobilized carbon black,but also the hue of the dyed or colored hair turned from bluish black toreddish black as illustrated in Table 15. In the table, the increasingnumber of the sign "+" indicates a higher degree of coloring. Inaddition, the bundle of human grey hair, which had been colored with ahair dye comprising anti-keratin antibody-immobilized carbon black,could not only be further richly colored with a hair dye comprisingrabbit anti-bovine IgG antibody-immobilized Titan Black, but also thehue of the dyed or colored hair turned from reddish black to bluishblack as illustrated in Table 15. Thus, it has been demonstrated thatrepeated colorings were effective in order to increase the degree ofcoloring of the hair that had been already colored in Example 12.

                  TABLE 15    ______________________________________            Primary   Secondary Degree of    Example Hair Dye  Hair Dye  coloring                                        Hue    ______________________________________    33      Example 2 Example 25                                +++     Reddish black            Example 2 --        ++      Bluish black    34      Example 5 Example 23                                +++     Bluish black            Example 5 --        ++      Reddish black    ______________________________________

Example 35 Rinse

The hair dye in Example 23 was compounded in a rinse according to amethod known in the art. The composition of the rinse having a coloringcapability is shown in Table 16.

                  TABLE 16    ______________________________________                        (% by weight)    ______________________________________    Stearyl trimethyl ammonium chloride                          3.0    Cetanol               1.5    Glycerine monostearate                          1.5    1,3-butylene glycol   5.0    Liquid paraffin       2.0    Perfume               proper quantity    Hair dye of Example 23                          5.0    Refined water         Balance    ______________________________________

Example 36 Rinse

The hair dye in Example 25 was compounded in a rinse according to amethod known in the art. The composition of the rinse having a coloringcapability is shown in Table 17.

                  TABLE 17    ______________________________________                        (% by weight)    ______________________________________    Stearyl trimethyl ammonium chloride                          3.0    Cetanol               1.5    Glycerine monostearate                          1.5    1,3-butylene glycol   5.0    Liquid paraffin       2.0    Perfume               proper quantity    Hair dye of Example 25                          5.0    Refined water         Balance    ______________________________________

Example 37 Rinse

The hair dye in Example 27 was compounded in a rinse according to amethod known in the art. The composition of the rinse having a coloringcapability is shown in Table 18.

                  TABLE 18    ______________________________________                        (% by weight)    ______________________________________    Stearyl trimethyl ammonium chloride                          3.0    Cetanol               1.5    Glycerine monostearate                          1.5    1,3-butylene glycol   5.0    Liquid paraffin       2.0    Perfume               proper quantity    Hair dye of Example 27                          5.0    Refined water         Balance    ______________________________________

Example 38 Multi-step Dyeing

After grey hair had been colored for a while with the rinse comprisingthe hair dye produced in Example 14, other rinses comprising the hairdyes produced in Examples 35,36 , or 37 were applied.

Test Example 18 Coloring Efficiency Enhanced by Multi-step Dyeing

As a result of the multi-step dyeing in Example 38, it has beenindicated that the rinse produced in Examples 35,36, or 37 could furtherincrease the degree of coloring. In the case of the rinse produced inExample 35, the hue of the dyed or colored grey hair turned from reddishblack to bluish black.

INDUSTRIAL APPLICABILITY

The present invention provides hair dyes that are excellent in coloringcapability, are resistant to color fading, do not stain skin because oftheir specific affinity to hair, do not irritate skin, and increasetactile properties of hair as well.

Further, in the case of hair cosmetics or hair care products containingconventional hair dyes, such hair dyes do not possess a specificaffinity to hair, and sometimes cause skin eruption, not to mention skinstains. Thus, because of these disadvantages, it has been impossible toincrease hair dye contents, thus requiring the repetition of hair dyeingoperation. In contrast therewith, the hair dye of this invention has anaffinity specific to hair so that the hair dyes can be added to haircosmetics or hair care products in sufficiently high concentration. Inaddition, the hair dye of this invention can be adapted for wideapplication in a variety of hair cosmetics or hair care products. Thus,there are practical and outstanding advantages such that, withoutproviding negative effects on the scalp, daily use of such haircosmetics of the present invention can maintain beautiful color, gloss,touch and tactile properties of hair.

We claim:
 1. A hair coloring composition comprising an antibody havingan immunoactivity against hair and a coloring material, wherein saidantibody is immobilized on said coloring material, said coloringmaterial is a composite of a macromolecular carrier and a coloringsubstance selected from the group consisting of dyes and pigments, andthe substance-constituting unit size of said coloring material is atleast equivalent to that of said antibody.
 2. A hair coloringcomposition as claimed in claim 1, wherein said coloring substance iswater-insoluble and a weight ratio of the antibody to the coloringsubstance ranges 1:5-1:100.
 3. A hair coloring composition as claimed inclaim 2, wherein said water-insoluble coloring substance is an inorganicpigment.
 4. A hair coloring composition as claimed in claim 1, wherein aweight ratio of the macromolecular carrier to the coloring substance insaid composite ranges 1:0.001-1:10.
 5. A hair coloring composition asclaimed in claim 4, wherein said macromolecular carrier iswater-insoluble.
 6. A hair coloring composition as claimed in claim 5,wherein said water-insoluble macromolecular carrier is composed ofparticulates having an average particle diameter ranging 0.001-100 μm,and 0.01-100 mg of said antibody are supported on 1 g of saidmacromolecular carrier.
 7. A hair coloring composition as claimed inclaim 6, wherein said water-insoluble macromolecular carrier has anaverage particle diameter ranging from 0.001-1 μm.
 8. A hair coloringcomposition as claimed in claim 7, wherein said water-insolublemacromolecular carrier has an average particle diameter ranging 0.05-0.7μm.
 9. A hair coloring composition as claimed in claim 4, wherein saidwater-insoluble macromolecular carrier is at least one compound selectedfrom the group consisting of water-insoluble polysaccharides,derivatives thereof, water-insoluble proteins, derivatives thereof,synthetic polymers and liposomes.
 10. A hair coloring composition asclaimed in claim 9, wherein said synthetic polymers are at least onepolymer having reactive groups selected from the group consisting ofpolystyrene, polychlorostyrene, polychloromethyl styrene, polyacrylicacid, polymethacrylic acid, polymaleic acid, polystyrene sulfonic acid,poly ( 2-acrylamide-2-methylpropane sulfonic acid),poly[N-2-hydroxypropyl) methacrylamide], poly(2-hydroxyethylmethacrylate), poly(glycerol monomethacrylate), poly(2-oxyethylacrylate), poly(2-oxyethyl methacrylate), polyethyleneglycolmethacrylate, copolymers thereof; and those polymers that have beenprovided with reactive groups by means of surface reforming.
 11. A haircoloring composition as claimed in claim 9, wherein said water-insolublepolysaccharides and derivatives thereof, and water-insoluble proteinscomprise at least one compound selected from the group consisting ofcross-linked, insolublized compounds and polymers of agarose, dextran,chitin, fibroin, gelatin and collagen.
 12. A hair coloring compositionas claimed in claim 4, wherein said macromolecular carrier iswater-soluble.
 13. A hair coloring composition as claimed in claim 12,wherein said water-soluble macromolecular carrier comprises at least onepolymeric compound selected from the group consisting ofpolysaccharides, proteins, derivatives thereof and synthetic polymers.14. A hair coloring composition as claimed in claim 12, wherein saidwater-soluble macromolecular carrier has a number-averaged molecularweight of 10,000-2,000,000, and 0.01-1,000 mg of the antibody aresupported on 1 g of said macromolecular carrier.
 15. A hair coloringcomposition as claimed in claim 1, wherein said coloring material iscomposed of a coloring substance having an average particle diameter of0.01-6 μm.
 16. A method of coloring hair, which comprises: treating hairwith a pretreatment agent for hair dye antibodies, said pretreatmentagent containing at least one chemical substance selected from the groupconsisting of reducing agents, surfactants, alkaline substances andenzymes; and then contacting the hair with the coloring composition ofclaim
 1. 17. A method of coloring hair, which comprises: contacting thehair with a hair coloring composition according to claim 1 followed bycontacting the hair with a hair dye comprising a coloring material onwhich a secondary antibody having an immunoactivity against saidanti-hair antibodies has been immobilized.
 18. A method of coloring hairwhich comprises: treating hair with a pretreatment agent for hair dyeantibodies, containing at least one chemical substance selected from thegroup consisting of reducing agents, surfactants, alkaline substancesand enzymes; and then contacting the hair with the hair coloringcomposition according to claim 1 followed by contacting the hair with adyeing agent comprising a coloring material on which a secondaryantibody having an immunoactivity against said anti-hair antibody hasbeen immobilized.
 19. A hair coloring composition as claimed in claim13, wherein said water-soluble macromolecular carrier has anumber-averaged molecular weight of 10,000-2,000,000, and 0.01-1,000 mgof the antibody are supported on 1 g of said macromolecular carrier. 20.A hair coloring composition comprising a coloring material and asecondary antibody having an immunoactivity against a first antibodyhaving an immunoactivity against hair , wherein said secondary antibodyis immobilized on said coloring material, said coloring material is acomposite of a macromolecular carrier and a coloring substance selectedfrom the group consisting of dyes and pigments, and thesubstance-constituting unit size of said coloring material is at leastequivalent to that of said secondary antibody.
 21. A hair coloringcomposition as claimed in claim 20, wherein said macromolecular carrieris water-soluble.